Most of the actions of cyclic AMP (cAMP) appear to be mediated via the activation of cAMP-dependent protein kinase. cAMP- dependent protein kinase phosphorylates and activates a protein phosphatase inhibitor, termed inhibitor-1, in skeletal muscle. The resulting inhibition of protein dephosphorylation would be predicted to further amplify the cellular response to hormones. The aim of this proposal is to utilize the current developments in molecular cloning to express the active fragment of inhibitor-1 initially in prokaryotic cells, and subsequently in eukaryotic cells to investigate its role in hormonal regulation. This will be achieved by construction of the synthetic gene for the 44 amino- acid minimal active fragment of rabbit skeletal muscle in E. coli. Biochemical characterization of the expressed peptide will be undertaken to establish its functional identity with inhibitor-1. The gene will be subcloned into both phagemid (for site-directed mutagenesis studies) and in eukaryotic vectors (to examine their effect on cellular regulation). The expression of the inhibitor-1 fragment in eukaryotic cells will be examined. Such studies will develop the probes necessary for the detailed investigation of the physiological role of protein phosphatase inhibitor-1 in mammalian cells.